Molecular phylogeny reveals cryptic diversity in Sibynophis from China (Serpentes: Sibynophiidae)

Abstract The elucidation of species diversity and distribution is critical within the fields of evolution, genetics, and conservation. The genus Sibynophis contains rare snakes that have historically received little attention. In this study, we conducted comprehensive sampling and use both mitochondrial and nuclear genetic markers to explore Sibynophis species diversity within China. Our findings revealed that S. c. miyiensis should be considered synonymous with S. c. grahami, and S. c. grahami should be gave a specific rank as S. grahami. In addition, we discovered S. triangularis was new to China and Myanmar. Based on the specimens and molecular phylogeny results, we redefined the species distribution boundaries of each Chinese species.

China (Günther, 1889), but is now known to occur in China, Korea, and Vietnam (Wallach et al., 2014). In China, the species exhibits broad distribution, ranging from southern Xizang in the west to Jiangsu in the east (Zhao, 2006). Three subspecies are currently recognized (Zhao, 2006;Zhao et al., 1998), including S. c. chinensis, S. c. grahami, and S. c. miyiensis. In addition to the nominal species, which broadly occurs in Southwest, Central, and East China,

S. c. miyiensis is endemic to Southwest Sichuan and Northwest
Yunnan, while S. c. grahami is endemic to northeastern Yunnan and Guizhou (Zhao et al., 1998;Zhao & Yang, 1997). This taxonomical arrangement is followed by most authors (Uetz et al., 2022;Wallach et al., 2014;Wang et al., 2020). The type locality of S. collaris is in Assam, India (Gray, 1853), but the species is restricted to Xizang and Yunnan in China (Zhao, 2006;Zhao et al., 1998).
Morphologically, the two species differ from one another by number of supralabials and anterior temporal scales only (Zhao, 2006;Zhao et al., 1998).
Due to their rare occurrence and elusive nature, few Sibynophis specimens have been collected since their initial description, and few studies have been conducted on their biology, particularly their systematics (Li et al., 2020;Pyron et al., 2013;Zaher et al., 2019). Although Sibynophis species have been included in certain phylogenetic studies (Chen et al., 2013;Li et al., 2020;Pyron et al., 2013;Zaher et al., 2019), only a limited number of samples have been examined. For example, Zaher et al. (2019) conducted a large-scale molecular phylogenetic study of advanced caenophidian snakes, which included five Sibynophis species, each with only one individual. Thus, given the small number of species and specimens studied, the diversity and evolution of the genus remain poorly understood.
Here, based on more extensive samples, we explored species diversity of the genus Sibynophis and revised the taxonomy and distribution boundaries of each taxon within China.

| MATERIAL S AND ME THODS
In total, 24 specimens previously identified as S. collaris and S. chinensis were collected from China (Figure 1), sequenced, and analyzed. Additional samples from the two species and their congeners were included, and their sequences were retrieved from GenBank (Table 1). Scaphiodontophis annulatus was chosen as the outgroup based on previous work .
Total DNA was extracted from 85% alcohol-preserved liver or muscle tissues using M5 HiPer Universal DNA Mini Kit (Mei5 Biotechnology Co., Ltd.) following the manufacturer's protocols.

| RE SULTS
The best-fit evolutionary models of the data were: HKY + G for the Uncorrected p-distances between species and clades are listed in

| DISCUSS ION
This study represents the first comprehensive investigation of

TA B L E 1 (Continued)
relationships reconstructed using the two methods and two datasets generated mostly identical topologies and highly similar support indices for major clades, with slight differences in support for  Unexpectedly, although clades A and D were highly supported, respectively, these two clades are not sister taxa that together form a monophyletic lineage (Figure 2). The genetic distance between the two clades was 12.6% (cyt. b-based), which exceeds that between certain species (e.g., 9.4% between S. subpunctatus and S. bistrigatus) ( Table 2). In addition, network analyses revealed that the two clades did not share any nDNA haplotype. These findings suggest that both clades represent two distinct species, which means S. c. grahami

| Systematics of S. chinensis
should be elevated to specific rank as S. grahami.
Given the taxonomic revision of S. chinensis (sensu lato), the distribution of S. grahami should be redefined accordingly. Thus, based on previous records (Zhao, 2006;Zhao et al., 1998) and our data, we conclude that S. grahami is present in southwestern Sichuan, Yunnan, and Guizhou in China (Figure 1).

F I G U R E 2
Bayesian consensus tree of Sibynophis inferred from mitochondrial DNA using models detailed in text. Posterior probabilities (>0.50) and bootstrap support values (>50%) are given adjacent to respective nodes for major nodes. Branch support indices are not given for shallow nodes to preserve clarity.

| Distribution of S. triangularis and S. collaris in China
Sibynophis triangularis was originally described based on a single specimen from Thailand (Taylor & Elbel, 1958). Although initially considered a subspecies of S. collaris, it was subsequently elevated to a separate species, that is, S. triangularis (Taylor, 1965). At present, this species is known to occur in Thailand and Cambodia (Stuart et al., 2006;Uetz et al., 2022;Wallach et al., 2014). However, our molecular phylogenetic reconstruction revealed that a particular sample (FMNH 263023, Cambodia), previously identified as S. triangularis (Stuart et al., 2006), formed a well-supported clade (C) with specimens from Vietnam,  Figure 2). These findings suggest that this specimen was misidentified and may represent an undescribed taxon.
Thus, further examination of this specimen is required. In addition, in a previous large-scale systematic study of Chinese snakes, Li et al. (2020) identified one specimen (CHS 244) from Honghe, southern Yunnan, China, as S. collaris, and two specimens (CHS 879 and CHS 880) from Motuo, Xizang, China, as S. chinensis. Our results showed the first specimen was nested within the clade A and the latter two positioned in the clade B. Thus, we concluded that these samples were misidentified, with the former being S. grahami and the latter two being S. collaris. Zhao and Yang (1997), Zhao et al. (1998), Zhao (2006, and Yang and Rao (2008) proposed that S. collaris is distributed in southern Xizang and northwestern Yunnan in China. Based on our molecular phylogenetic analyses, however, several specimens collected from northwestern Yunnan, which is geographically close to Motuo, Xizang, were identified as S. grahami and S. triangularis (Figure 2).
Thus, it is most like that S. collaris is endemic to Motuo in Xizang, with no occurrence in Yunnan.

F I G U R E 3
The network based on c-mos gene (lines between the haplotypes represent one mutational step).
China and Myanmar. In total, four species, S. chinensis, S. collaris, S.
grahami, and S. triangularis, are distributed in China. Consequently, we revised their distribution boundaries in China and re-examined previously misidentified specimens.

ACK N OWLED G M ENTS
We would like to thank Zhong GH, Xie YL, Li K for their assistance in samples collection, and to Zeng YM and Li L for their help in data analyses and map preparation.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All DNA sequences newly generated are deposited in Genbank (accession nos.: OQ981548-OQ981602).